As used herein, the term “High Pressure Liquid Chromatography” (HPLC) means chromatography performed under pressures of up to 4,000 psi. There has been interest in performing liquid chromatography at elevated pressures, such as —Very High Pressure Liquid Chromatography (VHPLC): 6,000-20,000 pounds per square inch (psi) and Ultra High Pressure Liquid Chromatography (UHPLC): 20,000-120,000 psi. At pressures greater than 6,000 psi, injection valves typically used in HPLC applications fail due to leakage.
When performing on-line operations with a liquid chromatography separation column, an apparatus needs to alternate between supplying solute and supplying sample to the column. Valves have been the primary means used to switch between the solute and the sample. One mechanism has been the rotary injector valve where solute is directed to the column for one position of the valve and solute is directed through a loop containing a sample for the other position of the valve, thus introducing the sample as a discrete plug in the flowing stream. This mechanism works well when the pressure needed to inject is less than 5,000 psi. However, the improved chromatographic resolution of columns packed with smaller diameter particles, which require higher pressures to move liquids through them, is moving the industry toward columns where an increased pressure is required. Hardening the valves associated with the rotary injector allows the rotary injector valve to operate at up to 15,000 psi, but there is excessive wear and the valves exhibit a shorter lifetime.
A method of using fluid within nano-scale capillaries and channels (Nano-scale capillaries and channels are those having an inner diameter less than 200μ) to act as an on/off valve by freezing and thawing that liquid is known in the art, see, for example, U.S. Pat. Nos. 6,159,744 and 5,795,788. It has been found that the flow of liquids can be stopped or diverted to a further channel or chamber by merely freezing and thawing the liquid contained within a nano-scale segment of tubing or channel. This flow-switching device, referred to as “freeze-thaw valving,” requires no moving parts and most importantly contributes no dead volume within the analytical system. Freeze-thaw valving systems are essentially on/off fluidic valves that operate on the fused silica capillaries typically used for fluid connections and columns in liquid chromatography involving pressures greater than 4,000 psi. While activation times for these valves can be in the subsecond range, they close more slowly (5 sec typical) than they open. Further information about free-thaw valving may be reviewed in Applicant's U.S. PCT applications PCT/US/03/28910 and U.S. Pat. No. 6,557,575 each of which is hereby expressly incorporated by reference in their entirety.
A further means to load a sample onto a column is a manual process that uses a high pressure vessel dedicated to the loading purpose. One end of a HPLC column is placed in the chamber of this vessel at ambient pressure. The chamber is filled with the sample and then the vessel is brought to a high pressure, which forces a portion of the sample onto the top of the column. The high pressure is maintained for the length of time necessary to load a known quantity of sample onto the column. The pressure is returned to ambient after the loading operation and the loaded column is then removed from the loading vessel. The loaded column may be manually installed on a separation apparatus immediately or stored and undergo separation at a later time. In the separation apparatus, a high pressure pump is connected directly to the column and forces an elutant through the column and toward the detector device, such as a mass spectrometer. This sequence of operations must be repeated for each sample to be analyzed.
There is a need to have an apparatus that can, in an automated fashion, deposit a sample on a column and run the separation through the column at high pressures.